1. Field of the Invention
The present invention relates in general to methods for production and administration of pharmaceutical compounds, and more particularly to, polyriboinosinic-polyribocytidylic acid stabilized with polylysine and carboxymethylcellulose (Poly-ICLC).
2. Background Information
The invention described and claimed herein comprises an improved method for producing large lots of final sterile Poly-ICLC suitable for clinical use with reduced toxicity at effective dose levels, and a method for using Poly-ICLC clinically to treat various conditions and to regulate genes.
U.S. Pat. No. 4,349,538 (Hilton B LEVY) describes the preparation and clinical use of Poly-ICLC. However, the high doses (>300 mcg/kg) described clinically by Levy were intended to induce interferon and proved to be toxic and largely ineffectual for treatment of human patients, to the extent that, after many attempts, the experimental clinical use of poly-ICLC was largely discontinued over a decade ago. Thus, over 20 years after it was first disclosed, poly-ICLC has yet to be approved by the US Food and Drug Administration for any therapeutic indication.
Polyinosinic-Polycytidylic acid stabilized with polylysine and carboxymethylcellulose (Poly-ICLC) is a synthetic complex of polyinosinic and polycytidylic acid (double-stranded RNA (dsRNA)), stabilized with polylysine and carboxymethyl cellulose that was used as an interferon inducer at high doses (up to 300 mcg/kg IV) in short-term cancer trials some years ago. This gave mixed results with moderate toxicity, and the use of Poly-ICLC was generally abandoned when recombinant interferons became available. However, lower dose (10 to 50 mcg/kg) poly-ICLC results in a broader host defense stimulation, and enhanced clinical activity with little or no toxicity. (Salazar, Levy et al. 1996)), (Ewel, Urba et al. 1992) (Levy and Bever 1988) (Levy and Salazar 1992) (Talmadge and Hartman 1985). (Maluish, Reid et al. 1985) There are at least four interrelated clinical actions of poly-ICLC, any of which (alone or in combination) might be responsible for its antitumor and antiviral activity. These are 1) its induction of interferon; 2) its broad immune enhancing effect; 3) its activation of specific enzymes, especially oligoadenylate synthetase (OAS) and the p68 protein kinase (PKR); and 4) its multidimensional gene regulatory actions.
Interferon Induction. While induction of interferon is one of the important mechanisms for the action of poly-ICLC, interferon alone does not appear to be sufficient treatment for many conditions. In addition, the levels of serum interferon induced by our doses of Poly-ICLC are relatively low and have not in the past been associated with antiviral or antitumor action when interferon is administered alone exogenously. Immune modulation: Low dose Poly-ICLC also has a direct immune enhancing action independent of IFN, including, T-cell and natural killer cell activation, dendritic cell activation, cytokine release (e.g. interferons alpha, beta, and gamma, interleukins, corticosteroids, and TNF), and a potent adjuvant effect with increased antibody response to antigen. (Levy and Bever 1988) The immunostimulatory effects of Poly-ICLC and the interferons are complex. However, preliminary laboratory results in our pilot study in brain tumor patients showed no clear relationship between tumor response and measurable serum interferon, TNF, IL2, IL6, or neopterin. (Salazar, Levy et al. 1996) The adjuvant effect of poly-ICLC has also been demonstrated in several systems. For example, administration of low doses of poly-ICLC along with swine flu vaccination in monkeys dramatically accelerates and increases HAI titres. (Stephen, Hilmas et al. 1977). The complex interactions of the dsRNAs and the interferons in this regard are still incompletely understood, yet this seemingly paradoxical dual role of poly-ICLC as an antiviral agent and immune enhancer is consistent with its function in establishing an immediate defense system against viral attack while at the same time permitting the establishment of long term immunity. Thus, in contrast to conventional antiviral agents such as cidofovir, poly-ICLC could represent an ideal adjuvant to live virus vaccines, especially those such as smallpox vaccine that carry significant morbidity related to uncontrolled vaccine virus proliferation. In contrast to vaccination, the protective effect of dsRNAs such as poly-ICLC is also expected to be much more rapid, since the antiviral state is established within hours.
“Catalytic” Action of Poly-ICLC: OAS and PKR
The third action of Poly-ICLC is a more direct antiviral and antineoplastic effect mediated by at least two interferon-inducible nuclear enzyme systems, the 2′5′ oligoadenylate synthetase (OAS) and the P1/eIF2a kinase, also known as the dsRNA dependent P68 protein kinase (PKR). (Katze 1992), (Jacobs and Langland 1996) DsRNA is not a normal component of mammalian cells, but is a byproduct of many viral infections. DsRNA induces an antiviral state in cells by functioning as an obligatory cofactor for OAS, which activates ribonuclease-L, as well as for the PKR, which inhibits initiation of protein synthesis, and for an aminotransferase that is less well studied. This may help explain the demonstrated preferential decrease of tumor protein synthesis in vivo by poly-ICLC. The OAS and PKR are very sensitive to dsRNA dose and structure (Minks, West et al. 1979). For example, simple, long chain dsRNA (as in poly-ICLC) is the most potent stimulator of OAS and PKR, while mismatched or irregular dsRNA can be inhibitory. Similarly, the PKR has both high and low affinity binding sites and is inhibited by too high a dose of dsRNA. (Galabru, Katze et al. 1989) Clinically, the OAS response is also maximal at a dose of about 30 mcg/kg Poly-ICLC, and is much diminished above 100 mcg/kg (M. Kende, N. Bernton, et al., Unpublished).
The inhibition of EFC2 glioma cells in vitro by interferon beta is also significantly associated with activation of both the OAS and PKR. Others have demonstrated that expression of a functionally defective mutant of the PKR results in malignant transformation in vitro, suggesting an important role for this enzyme in suppression of tumorigenesis. (Koromilas, Roy et al. 1992) Both PKR and poly-IC are now know to regulate the p53 tumor suppressor gene, which in turn is associated with the multiple malignancy Li-Fraumeni syndrome, which includes astrocytomas, sarcomas, lung, and breast cancers.
The clinical half-life of the OAS response to IM Poly-ICLC is about 2.5 days, suggesting an optimum dose schedule of two or three times per week (M. Kende, N. Bernton, et al., Unpublished). Patients treated with Poly-ICLC showed up to a 40-fold increase in serum OAS product in response to treatment at 10 to 20 mcg/kg, and a significant association of serum OAS with tumor response (p=0.03). Mediation of antitumor action by OAS and/or PKR activation could help explain why the high doses of Poly-ICLC used in early cancer trials were relatively ineffective.
Many viruses, including but not limited to adenovirus, pox viruses (vaccinia), foot and mouth virus, influenza, hepatitis, poliovirus, herpes simplex, SV-40, reovirus, and the human immunodeficiency virus (HIV) circumvent host defenses by down regulating OAS and/or PKR, and this effect can be reversed in vitro by exogenous dsRNA. (Jacobs and Langland 1996) A block of either PKR and/or OAS-mediated interferon action might also explain the variable response to interferons seen in both microbial and neoplastic disease. Certain viruses as well as neoplasms such as malignant gliomas may use this or a similar mechanism to circumvent host defenses and cause disease. Those diseases may thus be among the prime targets for clinical Poly-ICLC therapy using the method described herein that maximizes PKR activation.
Poly-ICLC has thus been demonstrated to have significant antiviral action against a broad variety of virus families. One example is the inhibition of vaccinia virus in several models. (Levy and Lvovsky 1978), (Burgasova 1977), (Worthington and Baron 1973) (Baron, Salazar et al. 2003) Levy & Lvovsky used poly-ICLC or placebo topical ointment in rabbits and subsequently inoculated them with intradermal injections of vaccinia virus in 10 adjacent skin sites. Local treatments were repeated at 1, 2, 3, and 4 days. Animals treated with placebo ointment developed severe lesions from days 3 to 6, and three of the eight died with vaccinia encephalitis. In contrast, poly-ICLC treated animals showed no signs of systemic disease and had much smaller skin lesions, rarely progressing beyond 1-3 mm. In separate experiments, poly-ICLC was also effective when applied after the lesions became visible. Viral titers in the skin lesions were markedly decreased (by 3 logs) in the treated animals, and interferon titers were increased. However, the mean virus-neutralizing antibody titers in the serum at 10 days was increased about 10-fold in the treated animals compared to placebo controls. While the authors appeared to suggest that the beneficial effects were due to local skin action of the poly-ICLC, they also demonstrated a robust systemic (serum) interferon response to the topical administration of the drug. This suggests that the principal protective effect may actually be systemic, which is further supported by the marked decrease or possible abrogation of systemic vaccinia dissemination by the topical poly-ICLC in their experiments.
The interaction of the type I interferons and poly-ICLC with each other in protection of the host from viral or neoplastic challenges remains unclear partly because of their overlapping functions. Nevertheless, the relationship of Poly-ICLC and the interferons can be manipulated to therapeutic advantage, At moderate to high doses, poly-ICLC is a powerful inducer of interferons, which in turn can induce synthesis of enzymes systems such as the OAS, PKR and others that themselves ultimately regulate specific protein synthesis. But, as noted above, the OAS, PKR, and likely others also require low-dose dsRNAs as obligatory cofactors to function, particularly if they have been blocked by viral and or neoplastic invasion. Low dose poly-ICLC is particularly effective clinically in this regard when administered in the regimen described in item 6 under ‘Summary of the Invention’ below.
Clinical Gene Regulation is a fourth mechanism by which Poly-ICLC can modify the biologic response and provide therapeutic benefit.
Plain, unstabilized poly-IC has been shown to up-regulate or down-regulate a broad variety of over 270 genes in cell culture (Geiss, Jin et al. 2001). However plain poly-IC is not effective in vivo in primates and many other species, and is of limited clinical utility. On the other hand, Poly-ICLC is expected to have broad gene regulatory actions when administered clinically in humans. These genes include but are not limited to the helicase, interferon induced protein (p56), tumor necrosis factor, interferon regulatory factor, matrix metalloproteinase, plasminogen activator, tumor protein p53, fibroblast growth factor, eukaryotic initiation factor 2, actin filament-associated protein, and VCAM-1. Some of these genes play critical roles in the body's natural defenses against a variety of neoplasms and microbial infections, and in controlling other cell functions, including protein synthesis, atherogenesis, programmed (apoptotic) cell death, cell metabolism, cellular growth, the cytoskeleton and the extracellular matrix. Gene activation is transient, lasting 24-48 hours, suggesting that repeated dosing at 2-3 day intervals will be necessary to achieve a therapeutic effect in some conditions. This is the schedule of administration that was used successfully in treatment of malignant gliomas and is further described below. (See below). For chronic or long term degenerative conditions administration may need to be extended for a period of years.
Prevention and Treatment of Ionizing Radiation Injury
Another action of dsRNAs and poly-ICLC in particular is its demonstrated protection from radiation injury. (Baze, Lvovsky et al. 1979), (Lvovsky, Levine et al. 1982) In one set of experiments, mice were treated with poly-ICLC intramuscularly at doses of 0.1 to 3 mg/kg before receiving an LD50 (30d) of ionizing radiation. Animals received either single or multiple treatments with PICLC at 8-72 hours prior to radiation exposure. Treated animals had a significantly increased survival, with a maximum dose reduction factor of 1.25. Thirty-day survival was increased by as much as 60% at a dose of about 700 Rads (From 33 to 93%). The time of maximum radioprotection did not coincide with induction of interferon, which occurred 24-48 hours earlier. This suggests that induction of enzymes such as the PKR and OAS may be more important to the radioprotective effect than simple induction of interferon. As noted above, the maximum OAS response after PICLC is about 48-72 hours after treatment with IM Poly-ICLC and coincides with the time of maximum radioprotection. Thus, a dosing schedule that maximizes not only OAS and PKR induction, but also their subsequent activation would promise an even greater radioprotective effect.
Data will be presented demonstrating the radioprotective effect of Poly-ICLC when given according to the double-dosing regimen described below that maximizes OAS and PKR activation.
Manufacture of Poly-ICLC
U.S. Pat. No. 4,349,538 (Hilton B LEVY) describes the preparation of 1-liter batches of poly-ICLC of specific composition yielding biologically active product.
However, in the preparation of large production batches (>30 liters) of poly-ICLC in accordance with good manufacturing practice (GMP), we encountered difficulties with the formulation of poly-ICLC that required critical modifications from the process described by Levy in order to yield large lots of final sterile product suitable for clinical use. Testing results indicate that those modifications resulted in a significantly improved and more biologically potent product than that described in U.S. Pat. No. 4,349,538.